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Intracellular Localization of Interleukin-6 in Eosinophils From Atopic
Asthmatics and Effects of Interferon g
By Paige Lacy, Francesca Levi-Schaffer, Salahaddin Mahmudi-Azer, Ben Bablitz, Stacey C. Hagen,
Juan Velazquez, A. Barry Kay, and Redwan Moqbel
Eosinophils, prominent cells in asthmatic inflammation, have
been shown to synthesize, store, and release an array of up
to 18 cytokines and growth factors, including interleukin-6
(IL-6). In this report, we show that IL-6 immunofluorescence
localizes to the matrix of the crystalloid granule in peripheral
blood eosinophils from atopic asthmatics using confocal
laser scanning microscopy (CLSM). Granule localization of
IL-6 was confirmed using dot-blot analysis and enzymelinked immunosorbent assay (ELISA) on subcellular fractions
of highly purified eosinophils produced from density centrifugation across a 0% to 45% Nycodenz gradient. IL-6 was
found to coelute with eosinophil crystalloid granule marker
proteins, including eosinophil peroxidase (EPO), major basic
protein (MBP), arylsulfatase B, and b-hexosaminidase. Immunoreactivity to IL-6 colocalized with granule-associated IL-2
and IL-5 in subfractionated eosinophils. We also made the
novel and compelling observation that interferon g (IFNg), a
Th1-type cytokine, stimulated an early elevation in eosinophil IL-6 immunoreactivity. A 2.5-fold enhancement of IL-6
immunoreactivity in eosinophil granules was observed within
10 minutes of IFNg treatment (500 U/mL), as determined by
subcellular fractionation and CLSM. These findings suggest
that IFNg has short-term effects on human eosinophil function and imply that a physiologic role exists for Th1-type
cytokine modulation of Th2-type responses in these cells.
r 1998 by The American Society of Hematology.
E
of IL-6 occur in synergy with other cytokines, principally IL-1,
IL-3, and GM-CSF. IL-6 has been shown to be a cofactor that
potentiates IgE production from switched B cells by enhancing
the effects of IL-4,10 suggesting a role for IL-6 in atopy and
Th2-type responses. Recent evidence suggests that IL-6 may be
involved in the pathophysiology of bronchial asthma.11,12 Elevated levels of circulating IL-6 were observed in asthmatic
subjects (both symptomatic and asymptomatic) compared with
normal controls. IL-6 levels were further increased during
natural exacerbation of asthma compared with asymptomatic
periods.11 Furthermore, bronchoalveolar lavage levels of IL-6
were found to increase after occupational allergen challenge.12
We have previously shown that mRNA encoding IL-6 and the
released product (by in situ hybridization and enzyme-linked
immunosorbent assay [ELISA], respectively) were upregulated
after treatment of highly purified human asthmatic eosinophils
with interferon g (IFNg).6 The effect of IFNg on stimulating the
release of other eosinophil-derived cytokines and chemokines
has also been observed.13-15 IFNg is a cytokine described as the
prominent product of Th1-type lymphocytes in both mouse and
human,16,17 and is usually associated with a wide range of
bacterial and viral infections. Serum levels of IFNg have been
shown to be elevated in acute severe asthma.18 Previous studies
have demonstrated that IFNg can influence human eosinophil
cytotoxicity and receptor expression following long-term (.12
hours) stimulation.19,20 The observation that IFNg, a Th1-type
cytokine, may be able to stimulate the release of a Th2-type
cytokine (IL-6) from human eosinophils suggests that a shift is
needed in our current appreciation of the relationship between
these two types of immune responses, at least at the level of the
eosinophil.
The aim of the present study is to determine the intracellular
site of storage for eosinophil-derived IL-6, and to analyze the
possible effects of IFNg on IL-6 mobilization within the
eosinophil. We hypothesized that (1) IL-6 is associated with the
matrix of the secretory granules in human eosinophils, and (2)
IL-6 storage and release in human eosinophils is regulated by
IFNg in a time-dependent manner. We tested these hypotheses
using a combination of in vitro assays, confocal laser scanning
microscopy (CLSM), and subcellular fractionation on peripheral blood eosinophils obtained from atopic asthmatic subjects.
OSINOPHILS, prominent cells in allergic inflammation
and asthma, have been shown to synthesize, store, and
release up to 18 inflammatory and regulatory cytokines and
growth factors,1 including interleukin-2 (IL-2), IL-4, IL-5, and
granulocyte/macrophage colony-stimulating factor (GM-CSF).2-5
We have previously reported that human asthmatic peripheral
blood eosinophils express mRNA for IL-6.6 In addition, IL-6–
positive eosinophils have been detected in blood from normal
donors, suggesting that IL-6 may be constitutively synthesized
and stored in unstimulated eosinophils.7 The site of storage of
IL-6 in eosinophils could not be determined by immunocytochemical staining, although it appeared to be associated with the
crystalloid secretory granule, because IL-6 immunoreactivity
showed a granular pattern of staining.6
IL-6 is a pleiotropic lymphokine shown to be released from a
wide range of tissue cell types, particularly fibroblasts, T cells,
and peripheral blood mononuclear cells.8,9 Its expression is
usually induced in cells by viral infection, lipopolysaccharide,
or other cytokines, depending on the cell type concerned. The
biologic effects of IL-6 range from stimulation of B-cell
hybridoma and mouse plasmacytoma growth (leading to enhanced monoclonal antibody production), B-cell terminal differentiation, T-cell proliferation, and induction of cytotoxicity, to
proliferation of hematopoietic progenitor cells.8,9 Many actions
From the Pulmonary Research Group, Department of Medicine,
University of Alberta, Edmonton, Alberta, Canada; and Department of
Allergy and Clinical Immunology, National Heart and Lung Institute,
London, UK.
Submitted July 28, 1997; accepted November 14, 1997.
Supported by The Wellcome Trust, UK; the Medical Research
Council, UK; Medical Research Council, Canada; and Alberta Heritage Foundation for Medical Research.
Address reprint requests to Redwan Moqbel, PhD, Pulmonary
Research Group, 574 Heritage Medical Research Center, University of
Alberta, Edmonton, Alberta, Canada T6G 2S2.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9107-0040$3.00/0
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Blood, Vol 91, No 7 (April 1), 1998: pp 2508-2516
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IL-6 IN EOSINOPHILS AND EFFECTS OF IFNg
We examined the colocalization of IL-6 with known granule
proteins in the presence or absence of IFNg stimulation. Our
results suggest an early effect of IFNg on eosinophil-derived
IL-6 storage and mobilization within the cell.
MATERIALS AND METHODS
Materials. Di-isopropyl fluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, Na-p-tosyl-L-arginine
methyl ester (TAME), 4-methylumbelliferyl sulfate, 4-methylumbelliferyl N-acetyl-b-D-glucosaminide, b-nicotinamide adenine dinucleotide (reduced form), Coomassie Blue G, and sodium pyruvate were
purchased from Sigma (Poole, Dorset, UK). Adenosine triphosphate
(ATP) was obtained from Boehringer (Mannheim, Germany). Nycodenz was purchased from Nycomed Pharma (Oslo, Norway) and from
GIBCO-BRL Life Technologies (Grand Island, NY).
Preparation of eosinophils. A sample of peripheral blood (100 mL)
was obtained from mild atopic asthmatic subjects displaying eosinophilia more than 10% and who were not receiving oral corticosteroids.
Red blood cells were removed by dextran sedimentation, with the
remaining cells subjected to density centrifugation on Ficoll to obtain a
granulocyte pellet. Eosinophils were then purified by immunomagnetic
selection using the MACS system (Becton Dickinson, Cowley, UK).
This method uses the expression of CD16 antigen on neutrophils,
because this antigen is absent from resting eosinophils, as previously
described.2,3,5,6,20,21 Briefly, anti-CD16 monoclonal antibody (MoAb)
bound to micromagnetic beads (Miltenyi Biotec, Bergisch-Gladbach,
Germany) was incubated with the granulocyte pellet for 40 minutes at
4°C. Contamination by mononuclear cells was avoided by coincubating
anti-CD14– and anti-CD3–coated micromagnetic beads (Lab Impex,
Teddington, Middlesex, UK; and Miltenyi Biotec). The mixture was
then passed through a ferrous matrix column held in the field of a
permanent magnet. Highly purified CD162 eosinophils (.99%) were
obtained by negative selection, depleted of the immunomagnetically
positive neutrophils (CD161).
Subcellular fractionation. Eosinophils were subjected to subcellular fractionation as previously described.2,5 Briefly, at least 5 3 107
purified eosinophils were treated with 2 mmol/L DFP, a serine protease
inhibitor, for 5 minutes at room temperature before sedimenting at
240g for 5 minutes. The pellet was resuspended in ice-cold 0.25 mol/L
Fig 1. Photomicrograph of a
human eosinophil detected in a
buffy coat cytospin. (a) The eosinophil was stained specifically
with mouse monoclonal anti–
human IL-6 using the APAAP
technique, showing a granular
pattern of immunoreactivity as
compared with the negative isotype control (b) (original magnification 3100).
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HEPES-buffered sucrose (containing 10 mmol/L HEPES, 1 mmol/L
EGTA, and 5 µg/mL each of leupeptin, aprotinin, and TAME, pH 7.4),
and the cells were centrifuged again at 4°C. Cells were resuspended in
homogenization buffer (HEPES-buffered sucrose supplemented with 2
mmol/L MgCl2 and 1 mmol/L ATP) to optimal subfractionating density,
between 10 and 15 3 106/mL, and subjected to 10 to 15 passes through
a ballbearing homogenizer (EMBL, Heidelberg, Germany) possessing
11 µm clearance. The homogenate was centrifuged at 400g for 10
minutes, and the resulting postnuclear supernatant was layered onto an
8-mL linear Nycodenz gradient (0% to 45% Nycodenz dissolved in
HEPES-buffered sucrose with protease inhibitor cocktail) in a Beckman
14 3 89-mm Ultra-Clear centrifuge tube (Beckman, High Wycombe,
UK). The postnuclear supernatant was subjected to equilibrium density
centrifugation at 100,000g for 1 hour at 4°C. Twenty-four 0.4-mL
fractions were collected from each preparation and stored at 4°C no
longer than overnight or 280°C until used. The density of each fraction
was calculated from its respective refractive index.
Marker enzyme assays. A total of four marker enzyme assays were
used for locating specific subcellular organelles to fractions collected
from density gradient centrifugation. Arylsulfatase B and b-hexosaminidase activities were measured in each fraction as markers for secretory
granules and lysosomes, using the method described by Levi-Schaffer et
al.5 Briefly, 50 µL diluted fraction (1:10 in HEPES-buffered sucrose)
was added to a black 96-well microplate and mixed with 50 µL
arylsulfatase B substrate solution (10 mmol/L 4-methylumbelliferyl
sulfate in 0.2 mol/L acetate buffer, 6 mmol/L lead acetate, and 0.1%
Triton X-100, pH 5.6) or 50 µL b-hexosaminidase substrate solution (1
mmol/L 4-methylumbelliferyl N-acetyl-b-D-glucosaminide in 0.2 mol/L
citrate buffer and 0.1% Triton X-100, pH 4.5) before incubating at 37°C
for 1 hour. The reaction was terminated by addition of 150 µL ice-cold
0.2 mol/L Tris, and the fluorescence was measured in a Titertek
Fluoroskan (Huntsville, AL) microplate reader (excitation 340 nm and
emission 450 nm). For marking the presence of secretory granules, we
measured eosinophil peroxidase (EPO) activity adapted from White et
al22 for microtiter plates, and in later experiments, tetramethylbenzidine
substrate solution ([TMB] Sigma, Oakville, Ontario, Canada) was used
as a safer substrate for EPO in place of the more commonly used
o-phenylenediamine HCl. Cytosolic activity was detected by assay of
lactate dehydrogenase (LDH) as previously described.5 Plasma mem-
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2510
brane activity was assessed by the dot-blot method with antibody to
CD9 as previously described.5 Protein measurements were made using
the Bradford dye-binding protein assay, with bovine serum albumin as
the standard.
Cytokine ELISA. IL-6 and IL-2 were assayed in fractions using
Quantikine ELISA kits (British Biotechnology, Oxford, England; and
R & D Systems, Minneapolis, MN). These assays have a detection
sensitivity of 0.08 and 6 pg/mL, respectively. Assays were performed in
duplicates (whenever possible) of undiluted fractionated material. IL-5
was quantified using an anti–human IL-5 MoAb as previously described.23 The detection sensitivity of the assay was 6.25 pg/mL.
Dot-blot analysis. This technique was used to confirm the presence
of IL-6 shown by ELISA and to detect the granule-associated proteins,
major basic protein (MBP) and eosinophil cationic protein (ECP), and
the eosinophil plasma membrane marker CD9. For detection of IL-6, an
anti–human IL-6 MoAb was used in parallel with a rabbit anti-mouse
antibody used as a negative control (British Biotechnology). MBP was
detected by an in-house mouse MoAb BMK-13 (supernatant IgG1) that
has been carefully validated, whereas ECP was detected using EG2
MoAb (supernatant IgG1; Pharmacia, Uppsala, Sweden). Anti-CD9
MoAb (purified IgG1) was purchased from Becton Dickinson. Both
anti-CD3 MoAb (supernatant IgG1) and anti-CD5 MoAb (purified
IgG1) were used as irrelevant negative controls for these MoAbs
(Becton Dickinson). A total of 2 µL of the supernatants were placed on
nitrocellulose strips, allowed to dry, and blocked in 5% milk powder
(Sigma). The blocked membrane strips were incubated with appropriate
antibodies, and after extensive washings in PBS-Tween 20, they were
incubated with biotinylated anti-mouse or anti-goat antibody followed
by streptavidin-alkaline phosphatase, washed, and developed. In the
IL-6 dot-blot assay, recombinant IL-6 was used as a positive control.
Fractional activities of the markers were assessed by staining density,
assigned arbitrary units, and converted to the percentage of total activity
for each specific protein.
Double labeling and CLSM. Cytospins of eosinophils (100 µL
0.5 3 106 cells/mL in RPMI supplemented with 20% FCS) were made
by vortexing slides in a Cytospin 2 (Shandon, UK) centrifuge (800 rpm
for 2 minutes) followed by fixation in 2% paraformaldehyde in PBS for
10 minutes. Slides were subjected to a wash step (five washes in
Tris-buffered saline [TBS]), followed by incubation in blocking solution (2% human IgG [Sigma Reagent Grade] in H2O) in a humidified
container for 1 hour. After a second wash step, TBS containing 1% rat
anti–human IL-6 MoAb (50 µg/mL) labeled with FITC (Pharmingen,
San Diego, CA) was added, and the slides were incubated for 1 hour.
Slides were subjected to a wash step again before adding 1% BMK-13
in TBS and incubating for 1 hour. Bound BMK-13 antibody was
detected by addition of 1% (50 µg/mL) Texas Red–labeled goat
anti-mouse antibody (Pharmingen) to washed slides and incubation for
1 hour. For comparison, 1% FITC-labeled rat IgG2a was used as an
isotype control (Pharmingen). After a final wash step, 10 µL antibleaching agent (0.4% n-propyl gallate [Sigma] in 3:1 glycerol:TBS) was
dropped onto each slide before cover slip attachment. Slides were
examined using a 1003 objective under a Leica confocal laser scanning
microscope (Heidelberg, Germany). This instrument contained a kryptonargon laser to allow simultaneous scanning of two excitation wavelengths (488 and 568 nm) so that two images could be acquired from a
single pass to minimize bleaching of the slides. Differences between the
photomultiplier tube sensitivities of the two fluorochrome emission
spectra were compensated during collection of the data to obtain images
of equivalent brightness. Images were stored on computer and transferred to Adobe Photoshop (Adobe Systems Inc, Mountain View, CA)
for cropping and sizing.
Data presentation. The bioactivity of eosinophil granule, membrane, and cytosol constituents after fractionation, including IL-6
quantitation by ELISA (picograms per milliliter) and dot blot, is
expressed as the frequency distribution as previously described,5 except
LACY ET AL
for the time course of IFNg stimulation, where IL-6 is quantitatively
displayed as picograms per fraction.
RESULTS
Immunocytochemistry using APAAP. Highly purified eosinophils from asthmatic subjects were examined using the APAAP
staining technique for the presence of IL-6 immunoreactivity.
Anti–IL-6 binding revealed cytoplasmic and/or granular staining of IL-6 in a subpopulation of cells (25% to 50%; Fig 1a),
suggesting that IL-6 may be stored inside the secretory granules, in the same manner as previously observed for other
eosinophil-derived cytokines.2,3,5 Staining of eosinophils with
an isotype control antibody was negative (Fig 1b).
Subcellular fractionation. Using subcellular fractionation
of eosinophils, we successfully separated some of the organelles
Fig 2. Average of profiles (from 4 patients) of arylsulfatase B,
b-hexosaminidase, protein, MBP, CD9, LDH, and EPO together with
IL-6 immunoreactivity as determined by ELISA and dot-blot analysis.
Measurements of activities were averaged and plotted as a function
of collected fractions.
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IL-6 IN EOSINOPHILS AND EFFECTS OF IFNg
in these cells, allowing analysis of organelle-specific protein
expression. The secretory granules, plasma membrane, and
cytosol were clearly resolved after density gradient centrifugation (Fig 2). Secretory granule activity was detected by assays
for EPO, ECP (Fig 3), and MBP, and was usually confined to a
single peak. We have previously shown that the pellet produced
from pooled fractions corresponding to peak granule protein
activity contains enriched secretory granules.5
The plasma membrane and cytosol detected by peak antiCD9 binding and LDH activity, respectively, sedimented at
densities much lower than those for secretory granules. Immunoreactivity for the plasma membrane antigen CD9 resolved
into two distinct peaks. The first peak, present in higher-density
fractions, sedimented in fractions containing maximal secretory
granule activity. The second peak of CD9 immunoreactivity,
which was much larger than the first, appeared at the expected
range of densities (1.04 to 1.17 g/mL) for plasma membranes.24
The protein assay showed enrichment of cellular protein in two
peaks across the gradient, corresponding to fractions containing
secretory granule activity and cytosolic activity. The cytosol, as
identified by the presence of LDH activity, was associated with
fractions of a density range of 1.03 to 1.07 g/mL, coeluting with
the second peak of total protein.
b-Hexosaminidase and arylsulfatase B activity, which coincided with that of EPO, consistently overlapped and usually
produced bisected peaks, suggesting that at least two subpopulations of secretory granules exist in eosinophils. Also displayed
in Fig 2 is the profile of IL-6 as measured by ELISA in a
representative sample, which peaked at or near the same density
(,1.2 g/mL) as MBP and EPO.
The sedimentation profile of IL-6 immunoreactivity by
ELISA and dot blot, along with that of ECP, EPO, and
b-hexosaminidase, for four separate fractionations are pre-
Fig 3. Four individual fractionations of human eosinophils
in which IL-6 immunoreactivity
is compared with b-hexosaminidase and EPO activities along
with ECP immunoreactivity (using EG2 MoAb in dot blot). (A to
C) IL-6 measured by ELISA; (D)
IL-6 measured by dot-blot analysis.
2511
sented in Fig 3. In one of four preparations, IL-6 immunoreactivity could be resolved as a single well-defined peak. Otherwise, two peaks of IL-6 immunoreactivity could be resolved on
the gradient in two preparations, whereas in the final preparation only a shoulder on a single peak was apparent (Fig 3).
These split peak profiles overlapped with those of bhexosaminidase and arylsulfatase B activities. In all cases, IL-6
immunoreactivity colocalized with fractions possessing peak
granule protein activity. Taken together, these results suggest
that IL-6 is associated with eosinophil secretory granules, and
may be present in more than one subpopulation of granules.
Using whole-cell samples, unstimulated eosinophils were found
to store an average of 25 6 6 pg IL-6/106 cells (n 5 4). We have
previously established that unstimulated eosinophils are able to
release an average of 190 6 18 pg/mL IL-6 in supernatants,
which increased to 403 6 214 pg/mL after 24 or 48 hours of
IFNg stimulation (106 cells per sample).6
Dot-blot analysis was used to confirm the results of our
ELISA for IL-6 (Fig 3D). All profiles of dot-blot analysis for
IL-6 showed two peaks of immunoreactivity, the first appearing
in fractions containing secretory granule activity and the second
present in cytosolic fractions as indicated by LDH activity. The
most likely explanation for the appearance of IL-6 immunoreactivity in the cytosolic fractions is granule breakage during
homogenization. In three of four cases, dot-blot results correlated with those in ELISA determinations. The fourth preparation of eosinophils yielded erratic values of IL-6 immunoreactivity by ELISA, although the dot-blot analysis for this sample was
similar to what was observed in the other three preparations
(Fig 3D).
Coelution of IL-2, IL-5, and IL-6 with granule markers. In
one preparation, IL-2, IL-5, and IL-6 immunoreactivities were
all determined by relevant specific ELISAs, and were detected
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2512
in the same fractions as those containing peak secretory granule
activity (Fig 4). This finding supports previous results showing
IL-5 immunoreactivity in the secretory granules of eosinophils.25
CLSM. In support of our findings using subcellular fractionation, unstimulated eosinophils showed colocalization of IL-6
and MBP immunoreactivity to the crystalloid granules using
CLSM in double-labeled cells (Fig 5A to C). Yellow regions in
combined fluorescent imaging correspond to overlapping green
and red images, and suggest that the two stained proteins reside
within the same intracellular compartment. Isotype controls
displayed minimal fluorescence background after subtraction of
autofluorescence (Fig 5D and E). Interestingly, anti–IL-6 fluorescence produced characteristic doughnut-like shapes corresponding to the granule matrix (Fig 5A, F, G, and K). Upon
increased magnification of combined images, these granular
shapes were found to possess red centers (crystallized MBP)
surrounded by green fluorescence, corresponding to the core
and matrix of the crystalloid granules, respectively (Fig 5F).
These observations suggest that eosinophil IL-6 is stored within
the matrix of the specific secretory granule.
Effects of IFNg stimulation. The effect of IFNg stimulation
was examined in subfractionations of unstimulated and stimulated eosinophils from the same donor at 10 minutes and 16
Fig 4. IL-2, IL-5, and IL-6 immunoreactivities in subcellular fractions of eosinophils shown in comparison with arylsulfatase B,
b-hexosaminidase, and EPO activities. (A) IL-2, IL-5, and IL-6 measured by ELISA; (B) marker enzyme assays for secretory granules.
LACY ET AL
hours of IFNg incubation. After 10 minutes of stimulation by
500 U/mL IFNg, immunoreactivity to IL-6 was elevated over
that of unstimulated cells (Fig 6), although it remained within
the secretory granule fractions. The granule-associated IL-6 was
reduced to prestimulation levels after 16 hours of incubation
with IFNg.
By confocal microscopy, 10 minutes of stimulation by IFNg
produced an apparent intensification of anti–IL-6 fluorescence
in the secretory granules (Fig 5G). Anti–IL-6 fluorescence
became diminished at 6 hours of stimulation (Fig 5H) and
dispersed throughout the cytoplasm after 18 hours of incubation
(Fig 5J). By 24 hours of IFNg stimulation, the morphology of
the cells deteriorated and staining became sporadic (data not
shown). During incubation with IFNg, IL-6 immunoreactivity
in CLSM results became progressively dissociated from that of
MBP in combined images, suggesting that IL-6 had mobilized
into separate intracellular compartments (Fig 5K to N). However, we were unable to confirm this finding in results from
subfractionation, which may have been a result of the very low
levels of IL-6 in the individual fractions.
DISCUSSION
In this study, we have shown for the first time that IL-6 is
localized to the matrix of secretory granules in eosinophils from
atopic asthmatics. We have based our findings on the comprehensive analysis of subcellular fractions of eosinophils, correlation
of cellular components with IL-6 immunoreactivity, and fluorescent–labeled cells using CLSM. The use of subcellular fractionation and CLSM combined provides a powerful tool for
determining the processes of storage and intracellular trafficking of important proteins in inflammatory cells. In subcellular
fractionation, maximal IL-6 immunoreactivity was found to
coelute specifically with granule protein markers. We have
previously shown that fractions containing eosinophil granule
proteins are concentrated in crystalloid secretory granules.2,3,5
IL-6 immunofluorescence was found to localize to the matrix of
the crystalloid granule in CLSM analysis of cytospin preparations of human eosinophils. Eosinophils are among the few
inflammatory cells capable of storing cytokines and chemokines
in their secretory granules. These stored products may be
rapidly mobilized from intracellular sites of storage and released locally during inflammation. Whether these proteins are
bioactive and exert their influence on the inflammatory milieu
in situ by paracrine, autocrine, or juxtacrine signaling remains
to be elucidated.
It is unlikely that IL-6 immunoreactivity detected in granulecontaining fractions results from internalized IL-6–bound receptors, because secretory granules sediment at a much greater
buoyant density than anticipated for endosomal structures.
These structures would contain any endocytosed ligand-bound
receptors, and would be expected to sediment at a density
equivalent to plasma membrane.24 Moreover, earlier results
have shown that eosinophils transcribe and translate the gene
encoding IL-6,6 implying that IL-6 is synthesized within the
eosinophil.
The significance of the association of IL-6 immunoreactivity
with secretory granules lies in the possibility that IL-6 may be
released by regulated exocytosis from the eosinophil. Eosinophils have been shown to undergo degranulation, measured by
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IL-6 IN EOSINOPHILS AND EFFECTS OF IFNg
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Fig 5. Confocal microscopy of double-labeled eosinophils. (A to C) Representative unstimulated eosinophils with the FITC channel
corresponding to IL-6 (A), the Texas Red channel corresponding to MBP (B), and the combined fluorescence (C). (D to E) Isotype controls for FITC
fluorescence and Texas Red fluorescence, respectively. (F) Close-up of granules from an unstimulated cell showing doughnut-shaped IL-6
immunoreactivity surrounding red centers of MBP immunoreactivity. (G to J) Time course of IFNg effects on IL-6 immunoreactivity in eosinophils
after (G) 10 minutes, (H) 6 hours, (I) 12 hours, and (J) 18 hours of stimulation by 500 U/mL IFNg. (K to N) Same time course as G to J, showing
combined images for IL-6 and MBP (original magnification 3100).
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2514
Fig 6. Eosinophils that were subfractionated to determine effects
of IFNg incubation (500 U/mL) on IL-6 concentration or localization.
Quantification of IL-6 in each fraction was made by ELISA. Marker
enzyme assays used in these experiments were EPO and bhexosaminidase. The experiments were conducted at different times
using purified blood eosinophils from the same donor. (A) Unstimulated eosinophils, followed by eosinophils stimulated for (B) 10
minutes and (C) 16 hours by IFNg. An average of 50 3 106 eosinophils
were used for each preparation.
the release of b-hexosaminidase and by patch-clamp analysis,
in response to intracellularly applied agonists.26,27 So far, the
mechanism of eosinophil-derived cytokine release has not been
investigated, although it is possible to evoke secretion of IL-6
by IFNg,6 suggesting that IL-6 is released by receptor-mediated
secretion.
We observed an early effect of IFNg on the intensification of
IL-6 immunoreactivity using two separate experimental approaches. This finding compelled us to reevaluate our current
appreciation of the short-term effects of this cytokine. By
subcellular fractionation of eosinophils and CLSM, a 2.5-fold
enhancement of IL-6 immunoreactivity was observed in eosinophil granules within 10 minutes of IFNg treatment. Such
short-term effects of IFNg on IL-6 mobilization within eosinophils from atopic asthmatics may occur in patients with acute
LACY ET AL
severe asthma. These patients exhibit high levels of serum
IFNg.18 Whether this is also relevant to viral exacerbation of
asthma remains to be established.28
The most plausible explanation for the observed short-term
effects of IFNg on IL-6 immunoreactivity is that IFNg may
activate a conformational change in the structure of IL-6 within
the granules. Alternatively, IFNg may stimulate the ‘‘unmasking’’ of putative prepro–IL-6, which is not recognized by the
MoAbs used in this investigation. The specificity of the
antibody for the prepro form of IL-6 as opposed to the secreted
form of this protein is not known. It is unlikely that de novo IL-6
synthesis and vesicular trafficking into the secretory granules
would occur within such a short time to account for the
enhanced IL-6 immunoreactivity in response to IFNg.
The CLSM results showing dissociation of IL-6 from MBPpositive granule cores during prolonged stimulation by IFNg
suggest that some form of translocation of IL-6 may occur
between the secretory granules and the plasma membrane, an
observation that we were unable to confirm with subcellular
fractionation. However, the sensitivity of detection of IL-6
immunoreactivity using CLSM is at least one order of magnitude greater than that of ELISA measurement of IL-6 activity in
subcellular fractions. Based on CLSM results, extragranular
anti–IL-6 staining may be related to (1) newly synthesized IL-6
in the Golgi and its transport to the secretory granules and/or (2)
a smaller class of secretory vesicles engaged in the process of
piecemeal degranulation. The latter process has been previously
suggested for physiological release of eosinophil granule proteins.29 The biosynthetic pathway of IL-6, as well as other
cytokines, in eosinophils remains unknown and is currently
being investigated by our laboratory.
The putative role of eosinophil-derived IL-6 remains the
subject of speculation in asthma and allergic inflammation. IL-6
has been shown to be associated with symptomatic and
asymptomatic asthma and with natural or induced exacerbations.11,12 The precise mechanisms involving IL-6 in asthma are
not clear, but it is likely that eosinophils are an important source
of this cytokine in asthmatic responses. IL-6 is an important
helper cytokine for primary antigen–dependent T-cell activation
and proliferation.30 Infiltration of CD41 T cells has been shown
to be an important component of the inflammatory response in
antigen-induced late-phase allergic reactions in human skin,31
lung,32 and nose.33 IL-6 participates in IL-4–dependent IgE
synthesis from B cells10 and acts in synergism with IL-3 and
GM-CSF to enhance the proliferation and growth of hematopoietic progenitors in humans.34,35 Furthermore, IL-6 is involved in
promoting secretion of IgA in mucosal tissue during inflammatory reactions,30 and secretory IgA has been shown to be a
potent trigger of eosinophil degranulation.36 In fibroblasts, IL-6
has been shown to inhibit cell growth,37 so the ‘‘repair’’ function
of these cells may be influenced by IL-6 during allergic
inflammation. IL-6 is released in association with protective
responses against infectious and parasitic agents,9 and this may
be related to the elevated eosinophil numbers seen in helminthinduced infection.38 In summary, eosinophils may serve as an
important cellular source of IL-6 in allergic and inflammatory
reactions.
Eosinophils also store a number of other cytokines in their
secretory granules, such as IL-2, IL-4, IL-5, and GM-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
IL-6 IN EOSINOPHILS AND EFFECTS OF IFNg
CSF.2,3,5,25,39,40 Our results showing expression and intracellular
localization of IL-5 to the specific secretory granules are in
agreement with other reports describing detection of IL-2 and
IL-5 mRNA and their related products in eosinophils from
patients with atopic asthma.2,39-41
Storage of cytokines may lend eosinophils the potential to
release these preformed regulatory proteins rapidly and allow
them to act locally during inflammation. In addition, eosinophilderived cytokines may perpetuate inflammatory responses and
prolong the survival of these cells with damaging sequelae.
Eosinophil synthesis and storage of cytokines, chemokines, and
growth factors in health and disease requires further investigation to determine how these cells contribute to the regulation of
allergic inflammation.
ACKNOWLEDGMENT
We would like to thank Dr David Huston and Richard Dickason
(Baylor College of Medicine, Houston, TX) for assistance in measuring
IL-5 in one of our subfractionations; Professor Bastien Gomperts
(University College, London, UK) for invaluable advice, support, and
supervision of P.L. during the early phase of this research; and Matthew
Wakelin and Janet North (National Heart and Lung Institute, London,
UK) for technical assistance.
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1998 91: 2508-2516
Intracellular Localization of Interleukin-6 in Eosinophils From Atopic
Asthmatics and Effects of Interferon γ
Paige Lacy, Francesca Levi-Schaffer, Salahaddin Mahmudi-Azer, Ben Bablitz, Stacey C. Hagen, Juan
Velazquez, A. Barry Kay and Redwan Moqbel
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